1. Field of the Invention
The present invention relates to microorganism strains capable of improving the production of recombinant proteins. The present invention also relates to processes for preparing such strains and to processes for producing recombinant proteins by employing the strains.
2. Background Art
The large-scale economic production of recombinant proteins is becoming increasingly important to the biotechnological and pharmaceutical industries. Generally, recombinant proteins are prepared either in mammalian cell cultures or in microbial systems. Microbial systems have the advantage over mammalian cell cultures in that it is possible to produce in this way recombinant proteins within a shorter period of time and at lower costs. The most common microbial organism for producing recombinant proteins is the bacterium E. coli. E. coli can in principle produce proteins in various ways:
1. intracellular production in the form of soluble protein;
2. intracellular production in the form of inclusion bodies;
3. secretion into the periplasm or the surrounding nutrient medium.
The complexity and costs of preparing the desired protein are also substantially determined by the costs of purifying the crude product to give said desired protein. These costs are in addition to the costs of producing the crude product which is present after fermentation in the form of a mixture comprising the recombinant protein and host proteins secreted naturally by the cell. The purification includes in most cases several stages and is carried out by means of chromatographic processes. In this connection, the removal of contaminating host proteins, some of which are immunogenic or toxic, plays an important part.
In E. coli, proteins are typically secreted via the “sec pathway”. To this end, the gene of the protein to be produced is linked to a signal sequence, resulting in a signal peptide-protein fusion being produced. The signal peptide mediates secretion of the protein through the cytoplasmic membrane into the periplasm via the endogenous bacterial sec system. In the process, the signal sequence is removed and the desired protein is obtained in the periplasm. The protein may then be purified from the periplasm. Under certain conditions or in certain bacterial strains, the protein is released from the periplasm into the surrounding nutrient medium (e.g. Ray et al. 2002; EP0338410B1; Nagahari et al. 1985; Yang et al., 1998) and may be purified from the latter.
Compared to other preparation processes, secretion offers the advantage of obtaining native, soluble, correctly folded protein which, when compared to the inclusion body process, need not be denatured and renatured—a step accompanied by high losses of yield. Moreover, the product obtained is contaminated with fewer host proteins compared to intracellular, soluble production, since the bacterial periplasm contains substantially fewer host proteins than the cytoplasm.
Secretion of the proteins into the surrounding nutrient medium offers the additional advantage of the protein in this case being present in an even purer form as compared to secretion into the periplasm. Moreover, the first purification step does not require any complicated preparation of the periplasm but rather requires a much simpler and more reproducible removal of whole cells.
The crude product in the preparation of proteins by secretion is contaminated with fewer host proteins over all than in intracellular production. Nevertheless, contaminating host proteins also play a part here, especially host proteins which are also naturally secreted by the bacterium and then located in the periplasm or in the outer membrane. These proteins are distinguished by the fact that their genes naturally include a signal sequence that mediates the secretion. Apart from the fact that these host proteins contaminate the crude product, they also compete with the protein to be produced for the components of the secretion apparatus, possibly resulting in a reduced secretion of the protein to be produced. However, the host proteins which cause contamination of the crude product fulfill a physiological role in the host cell. For example, these proteins may be involved in chemotaxis and special transport processes.
The literature describes that the production of proteolysis-sensitive, secreted proteins can be improved by deleting genes coding for periplasmic proteases. This has been described for degP, ompT, ptr, (U.S. Pat. No. 5,264,365; Baneyx & Georgiou, 1991; Wadensten et al., 1991). This effect can be attributed to eliminating the activity of the proteases which degrade the produced protein in the starting cell. Proteases endogenous to the host frequently degrade especially heterologously produced proteins in cells. However, the amount of contaminating proteases is negligible, since they are produced only in very low amounts in the host cell, due to their high activity and enzymic function. Thus, deleting these genes does not affect the degree of purity of the produced proteins.
WO 2004/035792 A1 describes the modification of certain host proteins, (e.g. PhoS/PstS, DppA, MBP, Trx) by mutations in individual amino acids, which alter the physical or biochemical properties (isoelectric point, hydrophobicity, size). This alteration of the physical or biochemical properties results in the resulting modified contaminating host proteins no longer being copurified with the desired produced protein in each case, since they behave differently on a chromatographic column, for example. The method cannot be utilized for producing any protein, since the contaminating host proteins have to be altered specifically for each protein to be produced because each protein has different biochemical properties. In the process according to WO 2004/035792 A1, production and functionality of the contaminating host proteins are retained despite their modification. Thus, the degree of purity of the crude product of the produced protein does not change but in each case removal of the contaminating host proteins from the protein is facilitated.
WO 98/18946 describes cells which, in addition to the protein to be produced, coexpress Dsb proteins and have a deletion in the wild-type pstS gene but at the same time express a pstS variant. Here too, the amount of contaminating host protein is thus unchanged.